Detecting anti-cholinesterase materials

ABSTRACT

A METHOD FOR DETECTING THE PRESSURE OF ANTICHOLINESTERASE MATERIALS WHEREIN A CELLULOSIC SHEET HAVING SULFITE POLYACROLEIN BOUNT CHLOINESTERASE DISPERSED THROUGHOUT THE INTERSTICES THEREOF IS WETTED WITH A LIQUID HAVING A PH OF 7.0-7.5 AND THE WETTED SHEET, AFTER EXPOSURE TO AIR OR WATER SUSPECTED OF CONTAINING ANTICHLOINESTERASE MATERALS, IS CONTACTED WITH A SOLUTION OF N-METHYL INDOXYL BUTYREATE AND THE PRESENCE OF THE ANTICHOLINESTERASE MATERIALS IS DETERMINED FROM THE OBSERVED COLOR.

United States Patent O 3,809,616 DETECTING ANTI-CHOLINESTERASE MATERIALS Edward Emil Schmitt and Richard Carl Capozza, Norwalk, Conn., assignors to American Cyanamid Company, Stamford, Conn. No Drawing. Filed Nov. 15, 1972, Ser. No. 306,780 Int. Cl. C12k 1/10 U.S. Cl. 195-103.5 R Claims ABSTRACT OF THE DISCLOSURE A method for detecting the presence of anticholinesterase materials wherein a cellulosic sheet having sulfited polyacrolein bound cholinesterase dispersed throughout the interstices thereof is wetted with a liquid having a pH of 7.0-7.5 and the wetted sheet, after exposure to air or water suspected of containing anticholinesterase materials, is contacted with a solution of N-methyl indoxyl butyrate and the presence of the anticholinesterase materials is determined from the observed color.

BACKGROUND OF THE INVENTION There has existed for a considerable period of time a need for an improved method for supporting horse serum cholinesterase in active and stable form. Present methods for accomplishing this end appear to produce stable products, however, enzyme migration and elution during contact thereof with liquids render them undesirable, if not useless.

These prior art systems generally constitute a fibrous mat produced from, for example glass fibers, having cholinesterase impregnated therein. These systems are generally used in the field in the detection of anticholinesterase materials e.g. nerve gas, which are fatal if inhaled or otherwise contacted by human beings and animals. The systems are almost always used in the form of kits which contain, in addition to the mat, a series of packaged liquid which must be used to wet the mat before the presence of anticholinesterase materials can be detected. In normal use, the cholinesterase impregnated glass mat is wetted with a standard buffer solution at a pH of about 7.0- 7.5 and the wet mat is then exposed to the suspected air or water and contacted with a solution of 2,6-dichloroindophenyl acetate. The wet spot is, at first, colorless. The cholinesterase of the mat hydrolyzes the ester and the free phenol oxidized rapidly results in the formation of a blue colored spot on the wetted mat thereby indicating the absence of anticholinesterase materials in the air or Water. When anticholinesterase materials are present in the suspected environment, however, they deactivate the cholinesterase and the wet spot remains colorless, thereby indicating the presence of such anticholinesterase materials.

Since the cholinesterase is impregnated into the glass fiber mat but not immobilized therein, when the mat is wetted, the cholinesterase tends to chromatograph toward 'the edges of the wet spot. This results in a non-uniform color development since the center of the wet spot then remains relatively colorless While the outer edges thereof turn blue in both the presence and the absence of anticholinesterase materials. The detection device may then be very difficult to analyze and leads to confusion and misinterpretation of results.

SUMMARY We have now discovered a new method for the detection of anticholinesterase materials and a new article of manufacture useful in said detection whereby uniform wetting by both the buffer solution and the substrate is 3,809,616 Patented May 7, 1974 accomplished and a uniform color change across the whole wetted area is achieved even at very low concentratrons of contained cholinesterase. Our method results in a very sensitive test for the detection of anticholinesterase materials, the necessary tools for the execution of which are easily packaged and extremely stable. The method gives a clear, positive test which is easily read and interpreted even b relatively inexperienced personnel.

DESCRIPTION OF THE INVENTION INCLUDING PREFERRED EMBODIMENTS As mentioned briefly above, our novel articles comprise a cellulosic sheet having sulfited polyacrolein bound chlorinesterase dispersed uniformly throughout the interstices thereof.

The sulfited polyacrolein having horse serum cholinesterase bound thereto forms no part of the instant invention per se and can be produced by sulfiting and crosslinking polyacrolein to render it hydrophilic. By hydrophilic is meant that the resultant polymer is swellable in or capable of taking up water but is not substantially soluble therein. The sulfited polyacrolein is water-insoluble but may take up and retain water such as by absorption, adsorption etc.

No criticality exists in the order of sulfiting and crosslinking the polyacrolein but binding the cholinesterase thereto should always be accom lished last. Sulfiting can be accomplished by treating the polyacrolein with a suitable sulfite or material which imparts a sulfite group onto the polymer chain such as a sulfite per se, a hydrosulfite, a bisulfite, sulfurous acid and the like. Specifically, alkali metal or alkaline earth metal compounds and other salts such as sodium, potassium, calcium, ammonium etc. sulfites, bisulfites, hydrosulfites etc. can be used. The reaction is generally run at 25-90" C. and under atmospheric pressure, a more precise method being disclosed in US. Pat. Nos. 2,657,192 and 3,271,334, which patents are hereby incorporated herein by reference. By the term sulfited, as used herein, is meant that the polyacrolein has been contacted with a sulfite so as to modify it and should not be construed to necessarily mean that any added sulfite groups per se remain on the polymer after cholinesterase binding, although such ma be the case.

After the sulfite treatment, the sulfited polyacrolein is immobilized e.g. insolubilized such as by chemical crosslinking with a cross-linking agent or an immobilization agent useful for this purpose. For example, the procedures of US. Pat. No. 3,459,710, incorporated herein by reference, can be followed. Using this method, the sulfited polyacrolein is contacted with, for example, a diamine such as ethylene diamine, tetramethylene diamine, N-methylethylene diamine, 1,6-hexamethylene diamine etc. at 0- 150 C. in a solvent.

Grafting of the sulfited polyacrolein can also be used to immobilize the polymer in addition to reacting it with such agents as 4-aminophenyl sulfide hydrochloride salt.

The cholinesterase is bound to the resultant hydrophilic, sulfited polyacrolein at a temperature below that at which it, the enzyme, is deactivated. Generally, temperatures below C., preferably 5-65 C. should be used. The binding is carried out in the presence of bulfers (pH 7.0-7.5) and with agitation. The binding is accomplished in the presence of water since organic solvents tend to inactivate the cholinesterase.

Typically, the bound cholinesterase material is dispersed throughout the interstices of the cellulosic web in amounts ranging from about 5.0% to about 50.0%, by weight, based on the weight of the web, the resultant sulfited polyacrolein containing paper having from about 0.1 to about 100.0 units, preferably, 0.390.0 units, of activity per /2 inch disc of normal paper sheet having a thickness of from about 3.0 mils to about 30.0 mils. Any

thickness of paper may, however, be used without detracting from the scope of the present invention.

Any fiber-forming cellulosic paper material may be used to form the novel articles of the present invention. The cellulosic paper may be made from all types of fiber stocks, including those of poor quality, such as oak, poplar, yellow birch and those of extremely short fiber length, as well as those of long fiber length and of good quality derivation, such as spruce and hemlock. A wide variety of fibrous cellulosic material used in the preparation of paper, board, and the like may be used such as kraft pulp, rag pulp, ground wood, sulfite pulp, alpha pulp and the like. Similarly, other forms of paper-forming fibrous cel lulose such as cotton linters, linen, and the like may be employed. These materials may be used alone or in admixture with fibers from other sources such as jute, hemp, sisal, strings, chopped canvas and other materials.

It is further stressed that the cellulosic paper may also be obtained from bleached or unbleached kraft, bleached or unbleached sulfite or bleached or unbleached semichemical pulps. In addition, the paper may be made from mixtures of cellulosic paper-forming pulps with up to of such other fibers and the like.-Filaments of cellulosic materials such as cellulose acetate, regenerated cellulose and the like may also be used.

For most purposes it is preferred that the starting cellulosic fibers be unsized and generally free of added resins. However, for some purposes, it may be desirable to employ as the paper base sheet, a porous, high wet strength paper such as may be obtained by incorporating into the paper from about 0.5 to 5.0%, by weight, based on the weight of the fibers, of a thermosetting aminoplast resin such as a urea-formaldehyde resin, a melamineformaldehyde resin and the like. Such wet strength cellulosic papers are obtained in the conventional way by the use of such a resin applied to the pulp suspensions.

The method of making the cellulose paper used in our invention is not critical and any known paper-making process may be employed, see, for instance, US. Pat. No. 3,551,205, hereby incorporated herein by reference. In the normal manufacture of paper, for example, cellulosic fibers, such as those derived from wood pulp, are beaten in water to disperse the fibers therein and to reduce them to a length and fineness suitable for paper making. During the beating operation the cellulosic fibers fibrillate to produce minute tendrils which serve to interlock the fibers together when they are deposited on the forming screen of the paper making machine from a slurry thereof to make a sheet therefrom as the suspending liquid is drawn through the screen.

The sulfited polyacrolein bound cholinesterase may be added to the cellulosic paper anytime during the production thereof or even after the paper has been sheeted as long as the temperature employed does not deactivate the enzyme, as mentioned above. Therefore, it is within the scope of the present invention to prepare a beater pulp of paper making cellulosic fibers of any convenient consistency. To this can be added the sulfited polyacrolein bound cholinesterase. The suspension is then agitated gently to distribute the material uniformly therethrough and the aqueous suspension is then sheeted, preferably at a pH of between 4.5 and 6.0, to form a wet, water-laid 'web containing the bound cholinesterase. The web is then dried, preferably in air or under vacuum. Vacuum drying with a desiccant of calcium chloride for 6 days has proven effective. Drying at elevated temperatures is to be avoided since high temperatures tend to deactivate the cholinesterase and render the dispersed material useless. In general, it is preferred that less than about 0.1% of residual water be retained in the final sheet.

By incorporating the bound cholinesterase within the paper as it is being formed, there is obtained by direct engagement of fibers and bound enzyme, an integral mechanical union between the bound enzyme and the paper. Excellent porosity and permeability of the paper permit circulation of the buffer and the substrate to be treated by the bound enzyme through the structure. Within the paper, the fiber components enmesh to form an integral sheet structure which provides a holding and reinforcing matrix for the bound enzyme dispersed therethrough.

In paper-tnaking mills where various pigments are added at the beater, the bound cholinesterase may be added therewith or at any point more than about one minute from the Wire. In mills where the pulp suspension is given heavy refining, the bound cholinesterase may be added to the heater to the refiner efiluent or to the screen effluent sufiiciently ahead of the wire so that deposition becomes substantially complete before sheeting. Thus the application of the bound cholinesterase may be easily adapted to most types of paper or mill conditions and may be added prior to sheet making or, less advantageously, may be applied to the sheet at a convenient point after sheet making, as discussed below.

Fibers Which are very slightly soluble in the slurry medium may also be used. For example, since the paper is generally cast from a water slurry, the fibers may include some proportion of slightly soluble polyvinyl alcohol fibers. In the paper, as the medium is removed, these fibers tend to adhere at fiber junctions thereby providing additional strength to the ultimate article. Furthermore, to provide additional porosity in the web, one may employ a second fiber which is insoluble in the slurry medium but which is selectively soluble in another medium with which the web may be subsequently washed to remove the soluble fibers thereby leaving additional void space.

Additionally, the bound cholinesterase may be incorporated into a sandwich of preformed sheets by first forming the sheet on a screen and then contacting the wet web with bound cholinesterase, preferably for a period of time sufficient to enable substantially complete dispersement of the bound enzyme bearing web and the entire structure can then be dried, as described above.

By employing the water-laying method, a cohesive paper sheet is obtained in most cases without need for any further binding material of the non-fibrous type. If a binder is found necesary, however, colloidal polytetrafluoroethylene aqueous emulsion may be added to the paper-making fibers and bound enzyme before the web is cast. Agitation of the slurry causes the colloidal suspension to be broken and the PTFE to be coagulated in the slurry. When the resultant slurry is cast, the PTFE extends throughout the web binding the bound cholinesterase with the fibers. Other polymeric materials can also be used for this purpose, as discussed briefly above.

In use, the cellulosic sheet having the sulfited polyacrolein bound cholinesterase dispersed throughout the interstices thereof will generally be packaged as a kit with at least two receptacles containing liquids with which the sheet is to be contacted. The sheet will normally be in the form of small discs /2 to 1" in diameter which may be placed beneath a hole in a covering sheet of material such as cardboard, plastic etc.

Accordingly to our novel method the sheet is then wetted with a buffer solution having a pH in the range of between 7.0 and 7.5. A preferred material found useful for this purpose is a solution of tris(hydroxymethyl) aminomethane adjusted to a pH of about 7.5 with 0.1 N hydrochloric acid. A 0.02 N standard phosphate buifer adjusted to a pH of about 7.5 with 0.1 N hydrochloric acid and water per se have also been found acceptable.

After the sheet is wetted with the buffer solution and exposed to air or water suspected of being contaminated with anticholinesterase materials, the wet sheet is then contacted with N-methyl indoxyl buty-rate. The butyrate, normally being a solid, is preferably used in the form of an anhydrous solution, the solvent of which is preferably a secondary or higher order alcohol such as isopropyl alcohol, Z-butanol, isoamyl alcohol, tert.-butyl alcohol, and the like or mixtures thereof. The use of any other solvent is tolerable, however, but we have found that the N-methyl indoxyl butyrate exhibits long term stability in these alcohols whereas its stability in other solvents is less satisfactory.

When the sheet containing the sulfited polyacrolein bound cholinesterase is wetted, its color ranges from a dark yellow to orange. The presence of any anticholinesterase materials in the vicinity of the wet sheet will then deactivate the bound cholinesterase. When the solution of N-methyl indoxyl butyrate is subsequently added, the sheet will remain yellow to orange and the subject conducting the test for anticholinesterase materials will know they are present. However, if no anticholinesterase materials are present, the addition of the N-methyl indoxyl materials are present, the addition of the N-methyl indoxyl butyrate solution will cause hydrolysis thereof by the active cholinesterase. The hydrolyzed butyrate is colorless and the color of the sheet remains the same. However, over a period of from about 1-2 minutes, the hydrolyzed substrate is oxidized by the oxygen in the atmosphere and the wet spot on the sheet becomes a deep green, thereby indicating to the observer that the immediate area is free of anticholinesterase materials such as nerve gas, sarin, prostigmine, eserine and the like.

Example A Solubilization of polyacrolein: To a suitable reaction vessel fitted with stirrer, condenser, thermometer, nitrogen gas inlet and constant temperature bath are added 344 parts of sodium metabisulfite and 2400 ml, of distilled water. The pH of this solution is adjusted to 5.6 with M sodium hydroxide solution and 300 parts of finely divided polyacrolein are added. The reaction is allowed to continue under a nitrogen blanket at 65 C. until a clear, viscous, water-soluble polyacrolein adduct forms. The reaction is cooled an stored.

Example B Cross-linking of soluble polyacrolein: To a suitable glass-lined reaction vessel equipped with stirrer and nitrogen gas inlet are added 2500 ml. of the polyacrolein bisulfite adduct produced in Example A, above, in 4000 ml. of distilled water. The solution is gently stirred and 300 parts of 1,6-hexamethylene diamine in 400 ml. of distilled water are added drop-wise over a 4 hour period. A yellow, cross-linked polymer becomes suspended and is heated to 60 C. under a heavy nitrogen blanket for 10 minutes and then cooled to room temperature. The polymer is filtered through cheese cloth, placed in a Buchner funnel and washed thoroughly with water. The crosslinked polymer is then slurried gently with ten times its volume of water for -20 minutes, allowed to stand minutes and filtered. The washing procedure is repeated until the pH of the washings are between 6.5 and 7.0. The solid adduct is then slurried gently for 20 minutes with 1.0 M disodium phosphate adjusted to pH 6.5 and Washed with distilled water.

The following examples are set forth for purposes of illustration only and are not to be construed as limitations on the present invention except as set forth in the appended claims. All parts and percentages are by weight unless otherwise specified.

Example 1 To a suitable reaction vessel is added 0.625 part of horse serum cholinesterase and 50 ml. of phosphate buffer (0.02 M; pH 7.4). The solution is allowed to stand in the refrigerator without agitation for 30 minutes. The enzyme is then completely dissolved by stirring. In a separate vessel are slurried parts of the modified polyacrolein of Example B, above (14 mg./g. binding capacity) with 50 ml. of the same phosphate buffer. After stirring 10 minutes, the pH is readjusted to 7.4 with 0.1 N sodium hydroxide. The contents of both vessels are then admixed and allowed to stir gently overnight at 40 C. The enzyme adduct is then filtered and washed with co- 6 pious amounts of deionized water. Consistent binding yields of 7597% are achieved using this method.

A 50/50 Albacel/Astracel pulp (concentration 2.6 g./ cc.) is washed with water and subsequently with methanol to remove any residual sulfite and is then dried. To 1.3 parts of the resultant pulp are added 5.0 parts of the wet enzyme adduct (100 units/ g.) in a suitable blending vessel. The ingredients are blended for 5 minutes, ice being added to the mixture to prevent heat build-up. The blended slurry is then processed into a paper mat about the thickness of standard filter paper and of six inch diameter on a British Hand Sheet Mold. The paper is dried in vacuo over a desiccant for 16 hours. The resultant dry sheet yields 236 /2" discs each with approximately 0.6 unit of activity.

One of the /2 inch discs is then wetted with phosphate buffer (pH 7.4) and is then contacted with a solution of N-methyl indoxyl butyrate in isopropanol and the color change observed. The results set forth below in Table I indicate the absence of anticholinesterase materials.

TABLE I Color: Total elapsed time Orange 0 min. 8.4 sec. L1ght green 1 min. 0.5 sec. Green 2 min. 0.4 sec. :Deep green 4 min. 3.8 sec.

Example 2 The procedure of Example 1 is again followed except that the buffer solution and N-methyl indoxyl butyrate are added to the disc in the presence of a gaseous anticholinesterase material. No color change occurs whatsoever on the disc after 10 minutes, i.e. the disc remains orange.

Example 3 The procedure of Example 1 is again followed except that 20% of Ti0 is added to the paper pulp. The color of the disc changes from pale yellow to pale green indicating the absence of anticholinesterase material.

Examples 4-8 The procedure of Example 1 is again followed except that the polyacrolein is solubilized with (4) sodium sulfite, (5) sodium hydrosulfite, (6) sulfurous acid, (7) calcium sulfite and (8) ammonium bisulfite. In each instance, a color change of orange to deep green is observed. No color change is observed when the procedure of Example 2 is followed on a second group of discs prepared with the same agents.

Examples 9-11 The procedure of Example 1 is again followed except the polyacrolein used is cross-linked with (9) methylene bisacrylamide; (l0) isophorone diamine and (11) 4-aminophenyl sulfide hydrochloride salt. Again a color change of orange to green is observed when buffer solution and N-methyl indoxyl butyrate are added to discs of the resultant paper in the absence of anticholinesterase material and no color change is observed when the two additives are added in the presence of a gaseous anticholinesterase material.

Example 12 The procedures of Examples 1 and 2 are again followed except a kraft pulp is used. Similar sharp changes in color and no color change are achieved when the resultant paper is treated in the absence and presence, respectively, of anticholinesterase material.

Example 13 Following the procedures of Examples 1 and 2 except that the wetting agent is water (pH 7.0), similar results are observed.

Example 14 The procedures of Examples 1 and 2 are again followed except that the N-methyl indoxyl butyrate is used as a solution in isoamyl alcohol. Similar results are obtained.

We claim:

1. A method for detecting the presence of anticholinesterase materials which comprises (A) wetting a dry cellulosic paper sheet having cholinesterase bound to a hydrophilic, sulfited polyacrolein resin physically dispersed throughout the interstices thereof with a liquid of a pH of from about 7.0 to about 7.5, (B) contacting the resultant Wet sheet with N-methyl indoxyl butyrate after exposure of the wet sheet to air or water suspected of containing such anticholinesteraase agents and (C) determining the presence of such anticholinesterase materials from the observed color of the resultant wet sheet.

2. A method according to claim 1 wherein said liquid is a buttered aqueous solution.

3. A method according to claim 1 wherein said N-methyl indoxyl butyrate is dissolved in a secondary or tertiary alcohol.

4. A method according to claim 3 wherein said secondary alcohol is isopropanol.

5. A method according to claim 3 wherein said alcohol is isoamyl alcohol.

6. A method according to claim 1 wherein said cellulosic sheet contains a Whitening agent.

7. An article of manufacture useful in the detection of 10 polyacrolein physically dispersed throughout the interstices thereof, (2) a liquid of pH of about 7.0-7.5 and (3) N-methyl indoxyl butyrate.

10. A kit according to claim 9 wherein (3) is a solution of N-methyl indoxyl butyrate in a secondary or tertiary 15 alcohol.

References Cited UNITED STATES PATENTS 3,730,841 5/1973 Salvatore et=al. 195Dig. 11X 3,689,224 9/1972 Agnew et a1. 195-103.5 R X 3,526,480 9/1970 Findl et a1.

OTHER REFERENCES Kay: Process Biochem, August 1968, pp. 36-39.

25 DAVIID M. NAFF, Primary Examiner US. Cl. X.R.

l95-63, 68, Dig. 11, 127; 23230 B, 253 TP 

